Nitrate Respiration of the Hyperthermophile Pyrobaculum aerophilum

小行星9631006 本项目的目的是研究极端高温条件下的反硝化机理。研究的重点将是反硝化第一步的酶硝酸还原酶。最适生长温度为100 ℃的超嗜热古菌将被作为模式系统进行研究。本项目的主要目标包括：（1）改进嗜气假单胞菌的细胞培养条件。 这将使用加压小瓶和/或补料分批发酵罐来完成，以防止硝酸盐呼吸过程中亚硝酸盐的毒性积累。 预计一旦更好地了解生物体的营养需求，细胞产量就可以增加。我们将尝试确定额外的碳源，电子供体，和微量元素，可以提高细胞产量。(2)硝酸还原酶的纯化和生理学表征将使人们了解高温酶的生物化学。除了已经存在的使用人工电子供体的酶测定之外，将开发涉及醌作为电子供体的其他酶测定来表征膜结合酶。将用洗涤剂从膜中提取酶，并使用适当的技术进行纯化。这种高温酶的生化特性将被检查：高温硝酸还原酶是含钼的酶，如已知的嗜温细菌硝酸还原酶？或者，它含有钨做其他金属酶从嗜热古菌？电子转移到酶的本质是什么？(3)为进一步了解硝酸还原酶基因的结构，调控和进化方面的资料，我们将对这些基因进行克隆和测序。将制备基因文库，并使用反向遗传学或PCR探针克隆nar基因。DNA序列应该揭示相当多的信息，以相关的嗜温对应物。这个研究项目将有助于我们了解极端嗜热生物的能量代谢，并扩大我们对能量代谢如何进化的认识。 %这个项目关注的是了解在非常高的温度下生长的被称为古细菌的不寻常的细菌。 嗜气热杆菌在100 ℃下生长最好，也可以在没有氧气的情况下使用硝酸盐代替生长。 在这项工作中，酶硝酸还原酶，利用硝酸盐的关键酶，将进行研究。 酶将被纯化，其性质将被确定。 这将提供有关高温下生命的新的和有趣的信息，并可能导致利用高温酶的生物技术应用。 ***

9631006 Schroder The objective of this project is to study the mechanism of denitrification under extremely thermophilic conditions. The focus of the research will be the enzyme of the first step of denitrification, nitrate reductase. The hyperthermophile archaeon which has an optimal growth temperature of 100 C will be Pyrobaculum aerophilum studied as the model system. The objectives of this project include: (1) the improvement of cell culture conditions for P. aerophilum. This will be done using pressurized vials and/or a fed-batch fermentor to prevent the toxic accumulation of nitrite during nitrate respiration. It is anticipated that the cell yields can be increased once the nutritional needs of the organism are better known. We will attempt to identify additional carbon sources, electron donors, and trace elements which may improve cell yields. (2) the purification and physiological characterization of the nitrate reductase will give insight into the biochemistry of a high temperature enzyme. Besides the already existing enzyme assay using artificial electron donor, additional enzyme assays involving quinones as electron donor will be developed to characterize the membrane-bound enzyme. The enzyme will be extracted from the membrane with detergents and purified using appropriate techniques. The biochemical properties of this high temperature enzyme will be examined: Is the high temperature nitrate reductase a molybdenum containing enzyme like the known mesophilic bacterial nitrate reductases? Alternatively, does it contain tungsten as do other metallo-enzymes from thermophilic archaea? What is the nature of electron transfer to the enzyme? (3) to gain further structural, regulatory and evolutionary information, the nitrate reductase genes will be cloned and sequenced. A gene library will be prepared and the nar genes will be cloned using reverse genetics or PCR probing. The DNA sequence should reveal considerable information as to the relatedness to mesophilic counterparts. This res earch project will contribute to our understanding of the energy metabolism in extremely thermophilic organisms and widen our knowledge as to how energy metabolism has evolved. %%% This project is concerned with understanding unusual bacteria known as archae that grow at very high temperatures. Pyrobaculum aerophilum grows best at 100 C and can also grow in the absence of oxygen using nitrate instead. In this work the enzyme nitrate reductase, a key enzyme for using nitrate, will be studied. The enzyme will be purified, and its properties will be determined. This will provide new and interesting information about life at high temperatures and may lead to biotechnology applications that make use of high temperature enzymes. ***